The present invention relates to novel water soluble, randomly substituted partial N-, partial O-acetylated chitosan or derivatives thereof, and preserving compositions containing water soluble, randomly substituted partial N-, partial O-acetylated chitosan, chitosan or derivatives thereof and novel processes for making water-soluble randomly substituted partial N-, partial O-acetylated chitosan, chitosan or derivatives thereof.
Ophthalmic products intended for repeated use after opening, that is xe2x80x9cmulti-dosexe2x80x9d products, must be preserved to minimize contamination with microorganisms during use. Preservatives that are used in ophthalmic solutions are often irritating to the eye, and at worst, may damage eye tissue after repeated use. Preservative problems may be worsened in contact lens solutions when a contact lens that has been exposed to a preservative in a lens care solution acts as a reservoir that concentrates the preservative in the eye.
In the United States, acceptably preserved pharmaceutical products, including ophthalmic, nasal and otic preparations, must achieve minimum performance standards when tested according to the procedures of the United States Pharmacopoeia Preservative Efficacy Test (PET). According to the PET protocol, adequately preserved formulations must reduce 0 day challenge inocula and 14 day re-challenge inocula of the bacteria Staphylococcus aureus, Pseudomonas aeruginosa and Escherichia coli by at least 99.99% (3 logs) within 14 and 28 days after the challenge date. In the fungal challenge portion of the PET, preserved formulations must not allow any growth of Aspergillus niger and Candida albicans within 14 and 28 days following the 0 day challenge. To demonstrate preservative efficacy for contact lens care products, a modified PET procedure is required by the FDA wherein a re-challenge of the test solutions is done on day 14 after the 14 day organism concentrations are determined.
Chitosan, the de-acetylation product of chitin, is a non-toxic biopolymer with weak antimicrobial activity. Heretofore, the use of chitosan to preserve pharmaceutical compositions has been hampered by its insolubility at pH above 6 and also because the antimicrobial activity of Chitosan in acidic solutions, by itself, is too low to meet PET requirements. Chitosan""s water solubility at near neutral pH can be improved by derivatization with hydrophilic functional groups, such as carboxymethyl or glycol substituents, or by selective N-acetylation of commercially available chitosans.
Considerable efforts have been made to extend the water solubility of chitosan at neutral pH. In Sannan et al., Makromol Chem. 177, 3589 (1976), it was reported that, by treatment of chitin with alkali under homogeneous conditions, chitin with about 50% deacetylation became water-soluble. However, long reaction time and large quantities of solvent are required in some stages, including neutralization of the reaction mixture and removal of the resulting salt. This laborious process would be troublesome especially in large-scale production.
Kurita et al., Carbohydrate Polymers 16, 83 (1991), also discloses preparing water-soluble chitosan with about 50% N-acetylation by acetylating a 90% deacetylated chitosan with a complex solvent system, comprising aqueous acetic acid/methanol/pyridine. Kurita et al. describes that the resultant partially N-acetylated chitosan is water soluble, if the degree of acetylation is controlled at 50% and the acetyl groups are distributed randomly. However, the huge excess of pyridine solvent used by the Kurita method made this process impractical. Furthermore, the reaction products have limited water solubility at neutral pH because heterogeneous reaction conditions were employed that restrict uniform, random acetylation. Specifically, Kurita""s chitosan reactant was not soluble in the reaction mixture, but instead it was dispersed as a swollen gel which hindered complete availability of reaction sites. In this case, the acetylation reaction would be favored in those chain segments that were most exposed and free to the reaction mixture, while other parts of the gel would be comparatively less acetylated due to steric interference from adjacent polymer chain segments. When taken as a whole, the polymer chain is not uniformly random, but instead is comprised of blocks of higher and lower acetylation.
Kubota et al., Polymer Journal. 29, 123 (1997), reported to have a facile preparation of water-soluble N-acetylated chitosan. In this reference, the chitosan is degraded by treatment with NaBO3, and the depolymerized product is then N-acetylated with acetic anhydride in aqueous acetic acid. Since both physical-chemical and biological properties of chitosan are dependent upon the chemistry of the polymer, such as the random distribution of a definite amount of acetyl groups and the molecular weight of the polymer, this process, which involves depolymerization, might alter the biological properties of chitosan.
The present invention is directed to a pharmaceutical preserving composition comprising: (a) at least one chitosan or chitosan derivative, and (b) at least one buffer solution.
The present invention is further directed to a method of preserving a contact lens solution, comprising mixing a contact lens solution with the composition comprising: (a) at least one chitosan or chitosan derivative, and (b) at least one buffer.
Moreover, the present invention relates to a method of disinfecting a contact lens, comprising soaking the contact lens with the composition comprising: (a) at least one chitosan or chitosan derivative, and (b) at least one buffer solution for a suitable period of time.
The present invention also is directed to a composition comprising (a) at least one chitosan or chitosan derivative, and (b) at least one buffer solution, wherein the at least one chitosan or chitosan derivative is prepared by a method comprising the steps of dissolving the at least one chitosan or chitosan derivative into an aqueous acidic solution and reacting the chitosan with an acetylating agent in the presence of a phase transfer reagent.
The present invention is further directed to a process for producing a water soluble, randomly substituted partial N-, partial O-acetylated chitosan or chitosan derivative, comprising the steps of dissolving a chitosan or chitosan derivative in an aqueous acidic solution and reacting the chitosan or chitosan derivative with an acetylating agent in the presence of a phase transfer reagent. In a further aspect, the invention relates to the product made by such a process.
The present invention is further directed to a water soluble, randomly substituted partial N-, partial O-acetylated chitosan or derivative thereof represented by the formula (I), 
wherein R1, R2 and R3 are independently H or C(O)CH3, wherein the chitosan or derivative thereof is partially acetylated such that R1 has a degree of substitution of C(O)CH3 of from about 24 to about 55%, and R2 has a degree of substitution of C(O)CH3 of from about 1 to about 60%, m is greater than 25, wherein the partial N-, partial O-acetylated chitosan or derivative thereof is randomly substituted and is water soluble.
In another aspect, the invention provides a pharmaceutical preserving composition comprising:
(a) at least one water soluble, randomly substituted partially N-, partial O-acetylated chitosan or derivative, of formula (I),
(b) and at least one buffer solution.
In yet another aspect, the invention provides a pharmaceutical preserving composition comprising the product formed from mixing components (a) and (b) as described in the above aspect.
In another aspect, the invention provides a phamaceutical preserving composition comprising:
(a) at least one water soluble, randomly substituted partial N-, partial O-acetylated chitosan or derivative,
(b) and at least one buffer solution,
wherein the at least one water soluble, randomly substituted partial N-, partial O-acetylated chitosan or chitosan derivative is prepared by a method comprising the step of reacting at least one randomly substituted partial N-, partial O-acetylated chitosan or chitosan derivative with a base in a solvent.
In another aspect, the invention provides a contact lens solution comprising the pharmaceutical preserving composition as described above.
In another aspect, the invention provides a contact lens solution comprising the product formed from mixing components (a) and (b) as described above.
In another aspect, the invention provides a process for producing a water soluble, randomly substituted partial N-, partial O-acetylated chitosan or chitosan derivative, comprising the step of reacting a randomly substituted partial N-, partial O-acetylated chitosan or chitosan derivative with a base in a solvent.
In another aspect, the invention provides a product produced by the method of reacting a water soluble, randomly substituted partial N-, partial O-acetylated chitosan or chitosan derivative with a base in a solvent.
In other aspects, the invention provides for products made by the processes of the invention.
Additional advantages of the invention will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention. The advantages of the invention will be realized and attained by means of the elements and combinations particularly pointed out in the appended claims. It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the invention, as claimed.
The present invention may be understood more readily by reference to the following detailed description of preferred embodiments of the invention and the Examples included therein.
Before the present compounds, compositions, articles, devices, and/or methods are disclosed and described, it is to be understood that this invention is not limited to specific synthetic methods, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting.
As used in the specification and the appended claims, the singular forms xe2x80x9ca,xe2x80x9d xe2x80x9canxe2x80x9d and xe2x80x9cthexe2x80x9d include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to xe2x80x9can acylxe2x80x9d includes mixtures of acyl groups, reference to xe2x80x9ca halogenxe2x80x9d includes mixtures of two or more such halogens, and the like.
Ranges may be expressed herein as from xe2x80x9caboutxe2x80x9d one particular value, and/or to xe2x80x9caboutxe2x80x9d another particular value. When such a range is expressed, another embodiment includes from the one particular value and/or to the other particular value. Similarly, when values are expressed as approximations, by use of the antecedent xe2x80x9cabout,xe2x80x9d it will be understood that the particular value forms another embodiment. It will be further understood that the endpoints of each of the ranges are significant both in relation to the other endpoint, and independently of the other endpoint.
In this specification and in the claims which follow, reference will be made to a number of terms which shall be defined to have the following meanings:
References in the specification and concluding claims to parts by weight, of a particular element or component in a composition or article, denotes the weight relationship between the element or component and any other elements or components in the composition or article for which a part by weight is expressed. Thus, in a compound containing 2 parts by weight of component X and 5 parts by weight component Y, X and Y are present at a weight ratio of 2:5, and are present in such ratio regardless of whether additional components are contained in the compound.
A weight percent of a component, unless specifically stated to the contrary, is based on the total weight of the formulation or composition in which the component is included.
xe2x80x9cAqueous acidic solutionxe2x80x9d means an aqueous solution having a pH below 7.0.
By the term xe2x80x9ceffective amountxe2x80x9d of a compound or property as provided herein is meant such amount as is capable of performing the function of the compound or property for which an effective amount is expressed. The exact amount required will vary from process to process, depending on recognized variables such as the compounds employed and the processing conditions observed. Thus, it is not possible to specify an exact xe2x80x9ceffective amount.xe2x80x9d However, an appropriate effective amount may be determined by one of ordinary skill in the art using only routine experimentation.
By xe2x80x9cpharmaceutically acceptablexe2x80x9d is meant a material that is not biologically or otherwise undesirable, i.e., the material may be administered to an individual without causing any undesirable biological effects or interacting in a deleterious manner with any of the other components of the pharmaceutical composition in which it is contained.
The term xe2x80x9cwater solublexe2x80x9d as used herein to describe the water soluble chitosans of the present invention, is meant to include chitosans or derivatives thereof having a water solubility of at least about 0.2% where solubility is measured by the test described for Examples 8-28 of the specification. Using this method, the water solubility of the randomly substituted partial N-, partial O-acetylated chitosan of the present invention is in one aspect at least 0.2%, in other aspects is up to 2%, and in other aspects (if greater than 0.200 g of chitosan is used in the test) is higher than 2%. Water solubilities of the chitosans of the present invention may be even higher than 2% or less than 0.2% when measured by other test methods. In such cases, the water solubility is dependent on the molecular weight of the polymer, the viscosity of the resulting aqueous chitosan solution and the conditions of the solubility test chosen.
By the term xe2x80x9crandomly substitutedxe2x80x9d is meant a random substitution of acetyl groups on the chitosan main chain, which contributes to the water solubility or hydrophilicity of the resultant chitosan polymer.
By the term water soluble, xe2x80x9cpartial N-, partial O-acetylated chitosanxe2x80x9d or derivative thereof is meant a poly(N-, O-acetylated-D-glucosamine).
By the term xe2x80x9cdegree of deacetylationxe2x80x9d is meant the percentage of free amino groups on the water soluble, chitosan or chitosan derivative. The percent of N-acetylation can be caluculated from the deacetylation value. The terms N-acetylation or O-acetylation are also referred to as the degree of substitution with C(O)CH3 on either N or O.
It is to be understood that greater than 50% N-acetylation is sometimes described in the art as a chitin. However, the term xe2x80x9cchitosanxe2x80x9d is used throughout the invention herein to include chitosans and, if the N-acetylation is greater than 50%, to include chitins.
By the term xe2x80x9cheterogeneous conditionsxe2x80x9d is meant that all or part of the reaction is carried out in a solid or highly swollen state, i.e., gel.
By the term xe2x80x9chomogenous conditionsxe2x80x9d is meant that the reaction is carried out completely in a solution.
The present invention is a preserving composition for pharmaceutical products. The preserving composition can be used in various ophthalmic products such as contact lens rinsing, lubricating, cleaning and storage solutions, artificial tear solutions and ophthalmic drugs. The compositions of the instant invention may also be used to preserve otic and nasal solutions.
Contact lens solutions in particular present a special preservative challenge because lens wearers are usually exposed to the preserving agents for many years on a daily basis. The possibility that the lens wearer can experience discomfort or develop sensitivity to the preservative is even higher than would be the case in short-term exposure. Typical contact lens solution preserving agents used in the prior art are sorbic acid, thimerosal, or DYMED(trademark) (polyaminopropyl biguanide).
The composition of this invention comprises at least one chitosan or chitosan derivative, and at least one buffer solution. The composition of this invention additionally may contain at least one biocidal adjuvant. Compositions of the present invention contain these components in amounts to be effective as pharmaceutical preserving compositions useful for preserving pharmaceutical products, including ophthalmic, nasal and otic preparations.
One preferred embodiment is used as a contact lens solution preservative. Another preferred embodiment is used as a contact lens disinfection regimen. When the composition comprised of at least one chitosan or chitosan derivative and at least one buffer solution is used in a method to preserve a contact lens solution, the contact lens solution is mixed with the composition. When the composition comprised of at least one chitosan or chitosan derivative and at least one buffer solution is used in a contact lens disinfection regimen, the contact lens is rinsed and rubbed with the composition, and the contact lens then soaks in the composition for a suitable period of time, such as not less than 15 minutes, more preferably for not less than 1 hour, even more preferably not less than four hours. Preferably, the soaking occurs at room temperature; however, any suitable temperature may be employed.
In a preferred embodiment, the chitosan and chitosan derivatives of the present invention have the additional advantage of being capable of performing several functions normally requiring other ingredients. For instance, in a preferred embodiment, the chitosan or chitosan derivative may, in addition to its preserving role, act as a natural surfactant, and aid in lens cleaning by emulsifying lens proteins and lipids away from the lens surface into solution. Furthermore, chitosan, as a polymeric saccharide, can be used in a preferred embodiment as a solution thickening agent and lens lubricant thereby enhancing lens comfort by reducing lens drying rate. As such, the chitosan or chitosan derivative in one embodiment of this invention has a demulcent effect so as to enhance lens wearer comfort.
Example chitosan or chitosan derivatives include chitosan salts, water-soluble chitosan, water-soluble, randomly substituted partial N-, partial O-acetylated chitosan, chitosan oligosaccharide, carboxymethyl chitosan, and hydroxyalkyl chitosan. The hydroxyalkyl substituents of the hydroxyalkyl, chitosans and the carboxymethyl substituents of the carboxymethyl chitosans could be attached to any of the pendant nitrogen or oxygen groups on the chitin or chitosan ring subunit. Specific preferred hydroxyalkyl chitosans include but are not limited to, hydroxyethyl chitosan (also known as glycol chitosan), hydroxypropyl chitosan, dihydroxypropyl chitosan, hydroxybutyl chitosan and dihydroxybutyl chitosan.
Example water soluble, randomly substituted partial N-, partial O-acetylated chitosan derivatives include such salt thereof, oligosaccharide thereof, carboxymethyl chitosan thereof, and hydroxyalkyl chitosan thereof. The hydroxyalkyl substituents of such hydroxyalkyl chitosans and the carboxymethyl substituents of such carboxymethyl chitosans could be attached to any of the pendant nitrogen or oxygen groups on the chitin or chitosan ring subunit. Specific preferred hydroxyalkyl chitosans of the partial N-, partial O-acetylated chitosan, include but are not limited to, hydroxyethyl chitosan (also known as glycol chitosan), hydroxypropyl chitosan, dihydroxypropyl chitosan, hydroxybutyl chitosan and dihydroxybutyl chitosan.
In an embodiment, a water soluble, randomly substituted partial N-, partial O-acetylated chitosan or derivative thereof represented by the following formula (I) 
wherein R1, R2 and R3 are independently H or C(O)CH3, wherein the chito san or derivative thereof is partially acetylated such that R1 has a degree of substitution of C(O)CH3 of from about 24 to about 55%, and R2 has a degree of substitution of C(O)CH3 of from about 1 to about 60%,
m is greater than 25,
wherein the partial N-, partial O-acetylated chitosan or derivative thereof is randomly substituted and is water soluble.
The term xe2x80x9cmxe2x80x9d is the number of repeat units in the water soluble, chitosan or polymer chain. In one aspect m is about 100,000, but in other aspects m can be higher. The molecular weight range of the water soluble chitosan or polymer chain herein refers to the weight average molecular weight. The weight average molecular weight of the water soluble chitosan or polymer is typically at least about 5,000. In one aspect the weight average molecular weight can be up to about 3,000,000, but in other aspects can be higher.
It is a separately surprising finding of one embodiment of this invention that chitosan or chitosan derivatives with certain buffer solutions such as borate or phosphate buffers, have higher antimicrobial activity as compared, for example, to similar formulations in citrate, and tromethamine (tris) buffers and in water. Thus, in one embodiment, the buffer solution may be comprised of a borate buffer. Suitable borate buffers include, but are not limited to, boric acid, sodium borate, potassium tetraborate, potassium metaborate, and mixtures of the same. In another embodiment, the buffer solution may be comprised of a phosphate buffer. Suitable phosphate buffers include, but are not limited to sodium dihydrogen phosphate and disodium hydrogen phosphate, and mixtures of the same.
The present invention includes a biocidal adjuvant. The biocidal adjuvant may be used against, for example, bacteria, fungi, and viruses. One advantage of the present invention is the surprising synergistic preservative effect of the composition. Suitable biocidal adjuvants include, but are not limited to, disodium ethylenediaminetetracetic acid (EDTA), nitrilotriacetic acid, and ethyleneglyco-bis(xcex2-amino-ethylether)-N,N-tetraacetic acid.
The present composition may contain several ingredients to perform the intended function of the composition. One possible additional component may be used to allow the composition to have an osmotic pressure near that of normal lachrymal fluids. Such a function may be achieved, for instance, by a tonicity agent, such as sodium chloride, potassium chloride or glycerol.
One feature of a preferred contact lens solution embodiment of the present invention is that proteins are stabilized against denaturing as compared to commercial multi-purpose contact lens solutions. In one embodiment, this effect may be accomplished by adding at least one surfactant to the composition. The surfactant may also aid in the cleaning of the lens. Typical surfactants include, but are not limited to, Pluronics(copyright) or poloxamers, which are block copolymers of ethylene oxide and propylene oxide, or Tetronics(copyright) or poloxamine, which are block copolymers resulting from addition of ethylene oxide and propylene oxide to ethylene diamine. Other surfactants that may be used in the invention include, but are not limited to, tyloxapol, octoxynols, nonoxynols, and Tweens(copyright) or polyoxyethylene sorbitan fatty acid esters.
The contact lens solutions of the present invention may, in another embodiment, contain viscosity agents to provide lubrication to the eye. Typical viscosity agents include polymeric saccharides such as dextran, cellulose derivatives such as carboxymethyl cellulose and hydroxypropyl methylcellulose, polyvinyl alcohol, polyvinylpyrrolidinone, polyethylene glycol, and glycerin.
The present compositions have at least minimal preserving activity. In one embodiment, the biocidal activity of the composition is sufficient to meet the performance criteria of the Preservative Efficacy Test (xe2x80x9cPETxe2x80x9d) of the USP (United States Pharmacopoeia) as modified by the FDA. As such, the present compositions reduce 0 day challenge inocula and 14 day re-challenge inocula of the bacteria Staphylococcus aureus (ATCC No. 6538), Pseudomonas aeruginosa (ATCC No. 9027) and Escherichia coli (ATCC No. 8739) by at least 99.99% (3 logs) within 14 days after the challenge and re-challenge dates, each. In the fungal challenge portion of the PET, the present composition does not allow any growth of Aspergillus niger (ATCC No. 16404) and Candida albicans (ATCC No. 10231) within 14 days following a 0 day challenge and a 14 day re-challenge. As such, the present invention may be used in a method of preserving a contact lens solution, wherein the contact lens solution is mixed with the composition.
In one embodiment, the composition of the present invention has a near neutral pH. This pH condition is preferred for compatibility with the organism, such as the human eye. As such, one preferred pH of the invention is from 6 to 8, preferably 6.6 to 7.8, and more preferably 6.8 to 7.2. Insofar as the antimicrobial activity alone of the composition is concerned, the lowest pH in the specified range is preferred. Given such preferred pH ranges, in one preferred embodiment, the chitosan or chitosan derivatives of the present invention are soluble at pharmaceutically acceptable pH levels. Another embodiment includes chitosan or chitosan derivatives that are near neutral soluble, meaning water soluble, from pH 6 to 8.
The chitosan and chitosan derivatives described in the present invention may be prepared by any method recognized in the art. Alternatively, in one preferred method, which is a method of one embodiment of the present invention, water-soluble, randomly substituted partial N-, partial O-acetylated chitosan and chitosan or chitosan derivative is prepared by dissolving the chitosan or chitosan derivative in an aqueous acidic solution and reacting the chitosan with an acetylating agent in the presence of at least one phase transfer reagent. The preparation of the water soluble, randomly substituted partial N-, partial O-acetylated chitosan or chitosan derivative thereof is carried out in a homogenous solution, which provides for the random acetylated substitution. The acetylating agent and phase transfer reagent(s) employed are used in an effective amount to be suitable for preparing the water-soluble, randomly substituted partial N-, partial O-acetylated chitosan and chitosan or chitosan derivative. In a preferred embodiment, the water soluble, randomly substituted partial N-, partial O-acetylated chitosan and chitosan preferably dissolves in solutions with near neutral pH values, such as from pH 6.0 to 8.0. Aqueous acidic solution refers to pH less than 7 and is typically the acidic pH used in the art for acetylation under heterogeneous conditions.
The acetylating agent acetylates the chitosan. As such, any known acetylating agent may be used. Example acetylating agents include, but are not limited to, acetyl halides, and acetic anhydride. A preferred acetylating agent is acetic anhydride.
The phase transfer reagent may be comprised of any phase transfer reagents known in the art. In general, the phase transfer reagent works across the water and organic phases. Suitable phase transfer reagents include, but are not limited to, those described in xe2x80x9cPhase-Transfer Catalysis,xe2x80x9d Starks, C., et.al. Chapman and Hall, 1994, which is incorporated by reference in its entirety. Example phase transfer reagents include, but are not limited to, quaternary ammonium salts(Eq. I), quaternary phosphonium salts (Eq. II), crown ethers (Eq. IIIa-IIIc), and pyridinium salts (Eq. IV).
[A]w[B]x[C]y[D]zN+Qxe2x80x83xe2x80x83(I) 
or 
[A]w[B]x[C]y[D]zP+Qxe2x88x92xe2x80x83xe2x80x83(II) 
where
each of w, x, y and z is an integer from 0 to 4 and w+x+y+z=4
Q is a counter-ion selected from Fxe2x88x92, Clxe2x88x92, Brxe2x88x92, Ixe2x88x92, CH3COOxe2x88x92, OHxe2x88x92, HSO4xe2x88x92, NO3xe2x88x92, PF6xe2x88x92, BF4xe2x88x92, HCOOxe2x88x92 and H2PO4xe2x88x92; and
A, B, C and D are each selected from C1-C18 alkyl, phenyl in which the phenyl ring is unsubstituted or substituted by C1-C8 alkyl, C1-C8 alkoxy, halo, hydroxy, phenoxy, nitro, carboxy, acetamido, or aryl, benzyl, cycloalkyl have 5-6 ring member or heterocyclic ring system.
In one preferred embodiment, quaternary ammonium salts (Eq. I) and quaternary phosphonium salts (II) include, but are not limited to, tetra C1-C4 alkyl ammonium halides, such as tetrabutylammonium bromide (xe2x80x9cTBABrxe2x80x9d), tetramethylammonium chloride (xe2x80x9cTMACIxe2x80x9d), tetrabutylammonium dihydrogen phosphate (xe2x80x9cTBADHPxe2x80x9d), and tetrabutyl ammonium iodide (xe2x80x9cTBAIxe2x80x9d); benzyl tri C1-C4 alkylammonium halides, such as benzyltriethylammonium chloride (xe2x80x9cBTEACIxe2x80x9d); and tetra C1-C18 phosphonium halides, such as tetrabutyl phosphonium bromide (xe2x80x9cTBPBrxe2x80x9d) and hexadecyltributyl phosphonium bromide (xe2x80x9cHDTRPBrxe2x80x9d).
A preferred embodiment includes a number of crown ethers (Eq. IIIa to IIIc) in practicing the present invention. 
where X=O or S, independently selected for each X
/=1 to 3
In one preferred embodiment, suitable crown ethers according to Eq. IIIa include, but are not limited to, 12-crown-4,15-crown-5,18-crown-6 and 1,4,7,10,13,16-hexathiacyclooctadecane. 
where m=1 to 3
In one preferred embodiment, suitable crown ethers in accordance with Eq. IIIB include, but are not limited to, benzo-12-crown-4, benzo-15-crown-5 and benzo-18-crown-6. 
where n=1 to 3
p=1 to 3
R3=H, C1 to C4 alkyl or halogen
In one preferred embodiment, example crown ethers suitable for Eq. IIIc include, but are not limited to, dicylohexano-18-crown-6, dicyclohexano-24-crown-8, dibenzo-18-crown-6, dibenzo-21-crown-7, dibenzo-24-crown-8, dibenzo-30-crown-10, di-tere-butyl-di-benzo-18-crown-6 and xe2x80x24-bromobenzo-18-crown-6.
Pyridinium salts (Eq. IV) may also be used in practicing the present invention. 
where R1=C1 to C18 alkyl, benzyl or carboxymethyl
R2=C1 to C4 alkyl, chloro, fluoro, bromo, hydroxy, C1 to C4 alkoxy or alkoxycarbonyl
X=counter ion of F, Cl, Br, I or p-toluene sulfonate.
Example pyridinium salts of Eq.IV include, but are not limited to, C1 to C18 alkyl pyridinium halides, such as 1-dodecylpyridinium chloride and 1-cetylpyridinium bromide, 1-benzyl pyridinium halides, and 1-benzyl-3-hydroxypyridinium chloride.
In another embodiment, which is a method of one embodiment of the present invention, the water-soluble chitosan or chitosan derivative is prepared by a method comprising the step of reacting at least one water soluble, randomly substituted partial N-, partial O-acetylated chitosan or chitosan derivative with a base in a solvent.
The base may be comprised of any bases known in the art. Example bases include, but are not limited to, alkaline hydroxides, such as potassium hydroxide or sodium hydroxide, and alkaline carbonates, such as sodium carbonate, or trisodium phosphate.
The solvent may be comprised of any solvent known in the art. Example solvents include, but are not limited to, alcohols, such as methanol, ethanol, or isopropanol, ethers such as diethyl ether or, tetrahydrofuran, polar solvents, such as dimethyformamide, dimethyl sulfoxide or, N-methyl pyrrolidinone and ketones such as acetone or 2-butanone.
This invention can be further illustrated by the following examples of various embodiments, although it should be understood that these examples are included merely for purposes of illustration and are not intended to limit the scope of the invention unless otherwise specifically indicated. The starting materials are commercially available unless otherwise described. All percentages are by weight unless otherwise described.